The objective of this proposal is to investigate the hypothesis that the transformation of putrescine to spermidine and spermine is primarily an evolutionary transition and that the temporal and spatial influences, i.e., when and where these substances are manufactured within the architectural framework of the cell, are likely to be predominant factors in determining the biological function of the individual amines. Three lines of investigation will be pursued. First, to investigate and develop the use of isolated cell suspensions and primay cultures of purified rat placenta trophoblasts as an experimental model for studying the role of putrescine in proliferation. Cells, prepared by collagenase digestion and separated by velocity sedimentation at unit gravity will be characterized by electron microscopy and biochemical reactivity with particular emphasis on the regulation of ornithine decarboxylase. Second, to perform a detailed kinetic analysis of polyamine metabolism in HeLa cells and L cells. These experiments will involve the use of labelled precursors such as ornithine, arginine and methionine and the quantitative determination of the production and conversion rates for each amine. The applicability of electron microscope autoradiography for determining the subcellular localization of the polyamines will also be explored. Third, the mechanisms of regulation of ornithine decarboxylase and S-adenosyl methionine decarboxylase will be investigated. Ornithine decarboxylase, purified from rat placental cell suspensions by column and affinity chromatography, and S-adenosyl methionine decarboxylase, purified from rat prostate, will be used to obtain antibodies in rabbits. These in turn will be used: (a) to develop immunoprecipitant methods for enzyme determinations, and (b) in the isolation of specific mRNA's so that enzyme regulation may eventually be studied in cell free systems. These studies will establish further the relationship between polyamines and the regulation of nucleic acid metabolism and will eventually contribute to our understanding of the differences between normal and uncontrolled growth.